Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in the Delaware River

Photosynthesis genes and operons of aerobic anoxygenic photosynthetic (AAP) bacteria have been examined in a variety of marine habitats, but genomic information about freshwater AAP bacteria is lacking. The goal of this study was to examine photosynthesis genes of AAP bacteria in the Delaware River. In a fosmid library, we found two clones bearing photosynthesis gene clusters with unique gene content and organization. Both clones contained 37 open reading frames, with most of those genes encoding known AAP bacterial proteins. The genes in one fosmid were most closely related to those of AAP bacteria in the Rhodobacter genus. The genes of the other clone were related to those of freshwater beta-proteobacteria. Both clones contained the acsF gene, which is required for aerobic bacteriochlorophyll synthesis, suggesting that these bacteria are not anaerobes. The beta-proteobacterial fosmid has the puf operon B-A-L-M-C and is the first example of an uncultured bacterium with this operon structure. The alpha-3-proteobacterial fosmid has a rare gene order (Q-B-A-L-M-X), previously observed only in the Rhodobacter genus. Phylogenetic analyses of photosynthesis genes revealed a possible freshwater cluster of AAP beta-proteobacteria. The data from both Delaware River clones suggest there are groups of freshwater or estuarine AAP bacteria distinct from those found in marine environments.     

Aerobic anoxygenic photosynthetic bacteria with zinc-bacteriochlorophyll

Naturally occurring chlorophyllous pigments, which function as the cofactor in the early photochemical reaction of photosynthesis, have been proven beyond question to be magnesium-complexed porphyrin derivatives. Phototrophic organisms that use (bacterio)chlorophylls ([B]Chls) containing metals other than Mg were unknown for a long time. This common knowledge of natural photosynthesis has recently been modified by the striking finding that a novel purple pigment, zinc-chelated-BChl (Zn-BChl) a, is present as the major and functional pigment in species of the genus Acidiphilium. Acidiphilium species are obligately acidophilic chemoorganotrophic bacteria that grow and produce photopigments only under aerobic conditions. Although the mechanism of photosynthesis with Zn-BChl a in Acidiphilium species is similar to that seen in common purple bacteria, some characteristic photosynthetic features of the acidophilic bacteria are also found. The discovery of natural photosynthesis with Zn-BChl has not only provided a new insight into our understanding of bacterial photosynthesis but also raised some interesting questions to be clarified. The major questions are why the acidophilic bacteria have selected Zn-BChl for their photosynthesis and how they synthesize Zn-BChl and express photosynthetic activity with it in their natural habitats. In this article we review the current knowledge of the biology of Acidiphilium as aerobic photosynthetic bacteria with Zn-BChl a and discuss the interesting topics noted above.     

Genome organisation of the marine Roseobacter clade member Marinovum algicola

The Roseobacter clade, belonging to the family Rhodobacteraceae of the class Alphaproteobacteria, is one of the major bacterial groups in marine environments. A remarkable wealth of diverse large plasmids has been detected in members of this lineage. Here, we analysed the genome structure and extrachromosomal DNA content of four strains of the roseobacter species Marinovum algicola by pulsed-field gel electrophoresis. They were originally isolated from toxic dinoflagellates and possess multireplicon genomes with sizes between 5.20 and 5.35 Mb. In addition to the single circular chromosomes (3.60–3.74 Mb), whose organisation seem to be conserved, 9 to 12 extrachromosomal replicons have been detected for each strain. This number is unprecedented for roseobacters and proposes a sophisticated regulation of replication and partitioning to ensure stable maintenance. The plasmid lengths range from 7 to 477 kb and our analyses document a circular conformation for all but one of them, which might represent a linear plasmid-like prophage. In striking contrast to other roseobacters, up to one-third of the genomic information (1.75 Mb) is plasmid borne in Marinovum algicola. The plasmid patterns of some strains are conspicuously different, indicating that recombination and conjugative gene transfer are dominant mechanisms for microevolution within the Roseobacter clade.     

True marine and halophilic anoxygenic phototrophic bacteria

Anoxygenic phototrophic bacteria are widely distributed in marine sediments and shallow waters of the coastal zone, where they often form intensely colored mass developments. The phototrophic bacteria have adapted to the whole spectrum of salt concentrations, from freshwater to saturated brines, and it is apparent that individual species have adapted well to particular habitats and mineral salts compositions, both qualitatively and quantitatively. This adaptation is reflected not only in the demand for defined ranges of salt concentrations, but also in the phylogenetic relationships of these bacteria, as established by 16S rDNA sequences. Major phylogenetic branches of purple sulfur bacteria are represented by: (1) marine and extremely halophilic Ectothiorhodospiraceae, (2) truly marine and halophilic Chromatiaceae and (3) freshwater Chromatiaceae, some of which are tolerant to low salt concentrations and are successful competitors in brackish and marine habitats. Quite similarly, salt-dependent green sulfur bacteria form distinct phylogenetic lines. In addition, also among the phototrophic alpha-Proteobacteria (purple nonsulfur bacteria), distinct phylogenetic lines of salt-dependent species are recognized. Available data give rise to the assumption that salt concentrations of natural habitats are an important selective factor that determines the development of a selected range of phototrophic bacteria in an exclusive way. As a consequence, the salt responses of these bacteria are reflected in their phylogenetic relationships.     

Comparative genomics and mutagenesis analyses of choline metabolism in the marine Roseobacter clade

Choline is ubiquitous in marine eukaryotes and appears to be widely distributed in surface marine waters; however, its metabolism by marine bacteria is poorly understood. Here, using comparative genomics and molecular genetic approaches, we reveal that the capacity for choline catabolism is widespread in marine heterotrophs of the marine Roseobacter clade (MRC). Using the model bacterium Ruegeria pomeroyi, we confirm that the betA, betB and betC genes, encoding choline dehydrogenase, betaine aldehyde dehydrogenase and choline sulfatase, respectively, are involved in choline metabolism. The betT gene, encoding an organic solute transporter, was essential for the rapid uptake of choline but not glycine betaine (GBT). Growth of choline and GBT as a sole carbon source resulted in the re‐mineralization of these nitrogen‐rich compounds into ammonium. Oxidation of the methyl groups from choline requires formyltetrahydrofolate synthetase encoded by fhs in R. pomeroyi, deletion of which resulted in incomplete degradation of GBT. We demonstrate that this was due to an imbalance in the supply of reducing equivalents required for choline catabolism, which can be alleviated by the addition of formate. Together, our results demonstrate that choline metabolism is ubiquitous in the MRC and reveal the role of Fhs in methyl group oxidation in R. pomeroyi.     

Dynamics of anoxygenic photosynthesis in an experimental green sulphur bacteria biofilm

The dynamics of sulphide oxidation in an experimental biofilm of the green sulphur bacterium, Prosthecochloris aestuarii , were studied using a newly developed light–dark cycling procedure. The biofilm was grown for 6 weeks in a benthic gradient chamber, in which gradients of light, sulphide and oxygen were imposed experimentally. The H₂S concentrations and pH were measured with microsensors as a function of depth in the biofilm and of time after a change in illumination status. The sulphide oxidation rates were calculated as a function of time and depth in the biofilm using a numerical procedure to solve the non-stationary general diffusion equation. A close agreement was found between the areal rates of anoxygenic photosynthesis during the cycling procedure and the steady state before the cycling experiment. For the different layers of the biofilm, the maximum activity was observed after 10–12 min of light exposure. After this maximum, sulphide oxidation decreased concomitantly with sulphide concentration, indicating sulphide limitation of anoxygenic photosynthesis. This lag time limits the application of the standard dark–light shift method with a brief light exposure of a few seconds and, therefore, the numerical procedure described in this study enables the depth distribution of anoxygenic photosynthesis rates in microbial mats to be determined more accurately.     

Genome sequence of the marine alphaproteobacterium HTCC2150, assigned to the Roseobacter clade

Here we announce the genome sequence of a marine bacterium, HTCC2150, that was isolated off the Oregon coast using dilution-to-extinction culturing and that is affiliated with the Roseobacter clade. The 16S rRNA phylogeny showed that the strain was closely related to members of the RCA clade. The genome sequence suggests that strain HTCC2150 is an organoheterotroph carrying diverse metabolic potential, including a close relationship with phytoplankton.     

On the photosynthetic properties of marine bacterium COL2P belonging to Roseobacter clade

Aerobic anoxygenic phototrophs (AAPs) are prokaryotic microorganisms capable of harvesting light using bacteriochlorophyll-based reaction centres. Marine AAP communities are generally dominated by species belonging to the Roseobacter clade. For this reason, we used marine Roseobacter-related strain COL2P as a model organism to characterize its photosynthetic apparatus, level of pigmentation and expression of photosynthetic complexes. This strain contained functional photosynthetic reaction centres with bacteriochlorophyll a and spheroidenone as the main light-harvesting pigments, but the expression of the photosynthetic apparatus was significantly reduced when compared to truly photoautotrophic species. Moreover, the absence of peripheral light-harvesting complexes largely reduced its light-harvesting capacity. The size of the photosynthetic unit was limited to 35.4 ± 1.0 BChl a molecules supplemented by the same number of spheroidenone molecules. The contribution of oxidative phosphorylation and photophosphorylation was analysed by respiration and fluorometric measurements. Our results indicate that even with a such reduced photosynthetic apparatus, photophosphorylation provides up to three times higher electron fluxes than aerobic respiration. These results suggest that light-derived energy can provide a substantial fraction of COL2P metabolic needs.     

Aerobic anoxygenic phototrophic bacteria in the Mid-Atlantic Bight and the North Pacific Gyre

The abundance of aerobic anoxygenic phototrophic (AAP) bacteria, cyanobacteria, and heterotrophs was examined in the Mid-Atlantic Bight and the central North Pacific Gyre using infrared fluorescence microscopy coupled with image analysis and flow cytometry. AAP bacteria comprised 5% to 16% of total prokaryotes in the Atlantic Ocean but only 5% or less in the Pacific Ocean. In the Atlantic, AAP bacterial abundance was as much as 2-fold higher than that of Prochlorococcus spp. and 10-fold higher than that of Synechococcus spp. In contrast, Prochlorococcus spp. outnumbered AAP bacteria 5- to 50-fold in the Pacific. In both oceans, subsurface abundance maxima occurred within the photic zone, and AAP bacteria were least abundant below the 1% light depth. The abundance of AAP bacteria rivaled some groups of strictly heterotrophic bacteria and was often higher than the abundance of known AAP bacterial genera (Erythrobacter and Roseobacter spp.). Concentrations of bacteriochlorophyll a (BChl a) were low ( approximately 1%) compared to those of chlorophyll a in the North Atlantic. Although the BChl a content of AAP bacteria per cell was typically 20- to 250-fold lower than the divinyl-chlorophyll a content of Prochlorococcus, the pigment content of AAP bacteria approached that of Prochlorococcus in shelf break water. Our results suggest that AAP bacteria can be quite abundant in some oceanic regimes and that their distribution in the water column is consistent with phototrophy.     

Comparative genomics of methylated amine utilization by marine Roseobacter clade bacteria and development of functional gene markers (tmm, gmaS)

The marine Roseobacter clade bacteria comprise up to 20% of the microbial community in coastal surface seawater. Marine Roseobacter clade bacteria are known to catalyse some important biogeochemical transformations in marine carbon and sulfur cycles. Using a comparative genomic approach, this study revealed that many marine Roseobacter clade bacteria have the genetic potential to utilize methylated amines (MAs) as alternative nitrogen sources. These MAs represent a significant pool of dissolved organic carbon and nitrogen in the marine environment. The marine Roseobacter clade bacterial genomes also encode full sets of genes providing them with the potential to generate energy from complete oxidation of the methyl groups of MAs. Representative species of the marine Roseobacter clade were tested and their abilities to use MAs are directly linked to the presence in their genomes of genes encoding key enzymes involved in MA metabolism, including trimethylamine monooxygenase (tmm) and gamma-glutamylmethylamide synthetase (gmaS). These two genes were chosen as functional markers for detecting MA-utilizing marine Roseobacter clade bacteria in the environment. PCR primers targeting these two genes were designed and used successfully to retrieve corresponding gene sequences from MA-utilizing isolates of the marine Roseobacter clade, as well as directly from DNA extracted from surface seawater obtained from Station L4 off the coast of Plymouth, UK. Taken together, the results suggest that MAs may serve as important nitrogen and possibly energy sources for marine Roseobacter clade bacteria, which helps to explain their global success in the marine environment.     

Genome sequence of strain HTCC2083, a novel member of the marine clade Roseobacter

Strain HTCC2083 was isolated from Oregon seawater using dilution-to-extinction culturing and represents a novel member of the Roseobacter clade. The draft genome sequence of HTCC2083 is presented here. The genome is predicted to contain genes for aerobic anoxygenic phototrophy, sulfite-oxidizing chemolithotrophy, anapleurotic CO(2) fixation, carbon monoxide oxidation, and dimethylsulfoniopropionate (DMSP) utilization.     

Aerobic Anoxygenic Photosynthesis in Roseobacter Clade Bacteria from Diverse Marine Habitats

The marine Roseobacter clade comprises several genera of marine bacteria related to the uncultured SAR83 cluster, the second most abundant marine picoplankton lineage. Cultivated representatives of this clade are physiologically heterogeneous, and only some have the capability for aerobic anoxygenic photosynthesis, a process of potentially great ecological importance in the world's oceans. In an attempt to correlate phylogeny with ecology, we investigated the diversity of Roseobacter clade strains from various marine habitats (water samples, biofilms, laminariae, diatoms, and dinoflagellate cultures) by using the 16S rRNA gene as a phylogenetic marker gene. The potential for aerobic anoxygenic photosynthesis was determined on the genetic level by PCR amplification and sequencing of the pufLM genes of the bacterial photosynthesis reaction center and on the physiological level by detection of bacteriochlorophyll (Bchl) a. A collection of ca. 1,000 marine isolates was screened for members of the marine Roseobacter clade by 16S rRNA gene-directed multiplex PCR and sequencing. The 42 Roseobacter clade isolates found tended to form habitat-specific subclusters. The pufLM genes were detected in two groups of strains from dinoflagellate cultures but in none of the other Roseobacter clade isolates. Strains within the first group (the DFL-12 cluster) also synthesized Bchl a. Strains within the second group (the DFL-35 cluster) formed a new species of Roseovarius and did not produce Bchl a under the conditions investigated here, thus demonstrating the importance of genetic methods for screening of cultivation-dependent metabolic traits. The pufL genes of the dinoflagellate isolates were phylogenetically closely related to pufL genes from Betaproteobacteria, confirming similar previous observations which have been interpreted as indications of gene transfer events.     

Possible quorum sensing in marine snow bacteria: production of acylated homoserine lactones by Roseobacter strains isolated from marine snow

We report here, for the first time, that bacteria associated with marine snow produce communication signals involved in quorum sensing in gram-negative bacteria. Four of 43 marine microorganisms isolated from marine snow were found to produce acylated homoserine lactones (AHLs) in well diffusion and thin-layer chromatographic assays based on the Agrobacterium tumefaciens reporter system. Three of the AHL-producing strains were identified by 16S ribosomal DNA gene sequence analysis as Roseobacter spp., and this is the first report of AHL production by these alpha-PROTEOBACTERIA: It is likely that AHLs in Roseobacter species and other marine snow bacteria govern phenotypic traits (biofilm formation, exoenzyme production, and antibiotic production) which are required mainly when the population reaches high densities, e.g., in the marine snow community.     

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.     

Ecology, inhibitory activity, and morphogenesis of a marine antagonistic bacterium belonging to the Roseobacter clade

Roseobacter strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of Roseobacter 27-4 is lethal to the larval pathogens Vibrio anguillarum and Vibrio splendidus in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of Roseobacter 27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures. Roseobacter 27-4 grew between 10 and 30 degrees C but died rapidly at 37 degrees C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37 degrees C in less than 2 days and at 25 degrees C in 8 days. Using Roseobacter 27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.     

Bacteriophytochromes in anoxygenic photosynthetic bacteria

Since the first discovery of a bacteriophytochrome in Rhodospirillum centenum, numerous bacteriophytochromes have been identified and characterized in other anoxygenic photosynthetic bacteria. This review is focused on the biochemical and biophysical properties of bacteriophytochromes with a special emphasis on their roles in the synthesis of the photosynthetic apparatus.